The studies proposed here will examine details of the spontaneous activation of pothrombin and factor X in blood by assaying from thrombin and factor Xa. Four major research objectives will be explored. 1) We will study thrombin activation of factors V and VIII by determining the stage in the clotting sequence when this occurs and how the rate and extent of prothrombin activation is subsequently influenced. These studies will be based on the determination of thrombin generation in blood by radioimmunoassay following various perturbations, e.g. addition of purified, thrombin-activated factor V or VIII; and by inhibition of the coagulation activity of these factors by specific antibodies. Molecular changes which result from thrombin-activation of factors V and VIII will also be studied, by preparing antibodies to the activated factors and examining immunochemical reactions with the native and activated factors. 2) We will develop a radioimmunoassay for human factor Xa. With this technique, I plan to study the spontaneous activation of factor X in blood and to determine the pattern and levels generated during clotting; and the temporal quantitative interrelationships between activation of factor X and prothrombin activation. We will also study the extent to which factor X activation is dependent on the availability of factor VIII and determine if the consumption of factor VIII limits additional factor X activation. 3) We will study the details of inactivation of spontaneously generated thrombin by antithrombin III in blood by either a radoimmunoassay for the thrombin-antithrombin III complex, or by radioimmunoelectrophoresis. Specifically, we wish to determine the kinetics, rate and extent of inactivation of thrombin by antithrombin III. Determination of the timing of the inactivation (i.e. immediately after clotting or some time later) may give us an idea as to the specificity and physiologic significance to this inhibitor. 4) Using the radioimmunoassay for thrombin and factor Xa, we plan to determine whether either circulates in a variety of thrombotic states and the influence of anticoagulant therapy on levels of these factors.